Responses to salinity in the littoral earthworm genus Pontodrilus

The cosmopolitan littoral earthworm Pontodrilus litoralis is distributed in tropical and sub-tropical coastal habitats, whereas P. longissimus is reported only in the Thai-Malay coastal line. In the present study, we examined the difference in salinity effect on the survival rate, wet weight (hereafter weight) change, behaviour, and osmolality of these two Pontodrilus species. A 28 d exposure to varying salinity concentration (0–50 ppt) revealed that P. litoralis is able to survive over a wide salinity range than P. longissimus, with the latter species exhibiting a low survival rate over the same salinity range. During short-term exposure (0–96 h) to a salinity of less than 30 ppt, P. litoralis exhibited weight gain and this was significant in the first 12 h of exposure. However, P. longissimus gained weight when exposed to salinity at under 10 ppt in the first 72 h of exposure. The two species of Pontodrilus behaved differently when exposed to different salinities. The coelomic fluid osmolarity of Pontodrilus was related to the exposure medium and was mostly maintained as hyperosmotic to the external medium over the range of salinities tested. This study shows how two different species of the littoral earthworm genus Pontodrilus respond to a change in salinity, which may explain their dispersal pattern and shape their distribution pattern throughout Southeast Asia.

www.nature.com/scientificreports/ and weight of Pontodrilus, and (ii) that, since P. litoralis is a cosmopolitan earthworm that occurs naturally in the sand surface areas where sharp salinity gradients are frequent along with a high salinity exposure, it will be able to survive wider salinity ranges and higher salinity concentration than P. longissimus, a species that inhabits a deeper level in the sand and preferentially occurs in estuaries.

Results
Field sampling and observation. A total of 15 habitats of Pontodrilus were observed, and the salinity level was recorded in the field (Fig. 1, Supplementary Table 1, Supplementary Fig. 1). Pontodrilus litoralis was found in various habitat types for example under trash or leaf litter on the sandy beach, in mangrove swamp, sanitary sewer emptying to a sandy beach, and estuaries. These sites included both undisturbed and disturbed areas. However, P. longissimus mostly occurred in estuaries. We observed six localities where P. litoralis was found co-existing with P. longissimus in estuary habitats. At these locations, P. litoralis was found under debris or trash in the topsoil while P. longissimus was found deeper than P. litoralis; at a depth of 10-50 cm. The highest salinity recorded in a P. litoralis habitat was 33 ppt, whereas it was 21 ppt for sites with P. longissimus.  weight change of littoral earthworms exposed for 28 d to different concentrations of salinity are presented in Fig. 2. Pontodrilus litoralis was able to survive a wide range of salinities (0-50 ppt) with the highest survival rate observed at ≤ 30 ppt. The survival rate then decreased as the salinity increased above 30 ppt, falling to 97.5% and 87.5% at 40ppt and 50 ppt, respectively. In contrast, the survival of P. longissimus was more strongly affected by changes in salinity with the highest survival rate recorded at 10ppt (ca. 97%), being lower at 0 ppt (ca. 56%), and decreasing at salinities above 10 ppt to ca. 76% at 20 ppt and 0% at 30-50 ppt. Relatively, P. litoralis have a higher survival rate than P. longissimus and is better adapted for survival over a wider salinity range. The weight change of P. litoralis was significantly affected by the salinity of the test medium, an increment in weight was observed when P. litoralis was exposed to a salinity of less than 40 ppt (highest increase as the salinity increased above 10 ppt to 40 ppt), but with a marked weight loss at 50 ppt. In contrast, P. longissimus exhibited a high weight loss at a low salinity of 0-20 ppt and was unable to survive at salinities above 20 ppt. These results clearly indicated that P. litoralis is better adapted than P. longissimus to changes in salinity, which would likely account for its wide distribution in different environments.
Short-term (0-96 h) exposure to different salinity concentrations. Pontodrilus litoralis exhibited the same weight gain pattern when exposed to a salinity of less than 30 ppt, where a significant weight gain was clearly observed over the first 12 h of exposure ( Fig. 3) followed by a sharp decrease in the weight gain and ending in a constant weight after nearly 96 h. A marked weight loss was observed when P. litoralis was exposed to a salinity of 40 and 50 ppt. For P. longissimus, there was an increment in weight over the first 72 h of exposure to a salinity of 0 ppt and 10 ppt, but a prolonged exposure (96 h) resulted in a significant weight loss.
Pontodrilus litoralis and P. longissimus behaved differently when exposed to different salinity levels; not all individual presented the same behavior patterns initially but towards the end of the exposure period (96 h), all of them showed similar patterns in the introduced test medium. At a salinity of 0-30 ppt, P. litoralis immediately coiled and knotted (Fig. 4A) its posterior end. This response was maintained for up to 24 h of exposure and then they started to relax and loosen the knot by fully stretching their body (Fig. 4B) and maintained a calm state with slow movement until the end of the experiment. Increasing the medium's salinity resulted in an increased body movement in P. litoralis with the most active was recorded at a salinity of 30 ppt. At 40 ppt, P. litoralis started to loosen its knot and stretch its body after 6 h of exposure, and the posterior end turned reddish (Fig. 4C), but they still moved a moderate amount until the end of the experiment. At 50 ppt, P. litoralis was inactive and became thinner at the posterior end (Fig. 4D). Besides these behavioral changes, the size changed significantly when P. litoralis was exposed to a high or a low salinity ( Supplementary Fig. 2). In contrast, P. longissimus reacted differently compared to P. litoralis. At a salinity of 0-20 ppt, slow movement and coiled at posterior end was observed in P. longissimus. After 12 h of exposure, they started to relax and loosen the knot by fully stretching their body until the end of the experiment. At 30 ppt, P. longissimus coiled and knotted its posterior end from the beginning of exposure until towards 96 h of exposure. At high salinities (40-50 ppt), P. logissimus immediately coiled and knotted its body with rapid movement, after 3 h of exposure P. longissimus was inactive and the whole body of the earthworm turned reddish and became thinner with 100% mortality was observed in all replicates of treatment. Further increased the salinity of the test medium to 40 (1309-1825 mOsm kg −1 , mean = 1465 mOsm kg −1 ) or 50 ppt (1557-2118 mOsm kg −1 , mean = 1748 mOsm kg −1 ) resulted in the coelomic fluid osmolarity becoming isotonic after 48 h of exposure, but the worms started to die if immersed in either of these salinities for more than 48 h. Pontodrilus longissimus showed a similar trend when it was placed in a 0 ppt salinity test medium (5-10 mOsm kg −1 , mean = 6 mOsm kg −1 ), it remained hypertonic (505 mOsm kg −1 ) to the medium (Fig. 6). When they were placed into medium with a salinity of 20 ppt (560-677 mOsm kg −1 , mean = 608 mOsm kg −1 ) or 30 ppt (912-1030 mOsm kg −1 , mean = 960 mOsm kg −1 ), the coelomic fluid of P. longissimus becoming isotonic after 48 h of exposure. However, at a salinity of 40 ppt (1315-1649 mOsm kg −1 , mean = 1413 mOsm kg −1 ), P. longissimus was not able to survive for more than 48 h of exposure.

Discussion
The established cosmopolitan littoral earthworm Pontodrilus litoralis was found to live in various types of habitats whereas P. longissimus was found only in estuaries with muddy sand. Sympatry of these two Pontodrilus species occurred in some localities, where P. litoralis was repeatedly found in the sand surface, while P. longissimus was found at deeper levels below the surface than P. litoralis 6,17 . Pontodrilus litoralis has been reported as a bioluminescence earthworm and uses this to avoid predation by littoral predators because P. litoralis live on the sand surface [17][18][19][20] . We suggest that salinity is one of the key factors affecting the distribution and habitat preference of Pontodrilus in this region. Congeneric P. longissimus inhabited mostly estuaries with a lower salinity compared to P. litoralis. www.nature.com/scientificreports/ The 28-d exposure to different salinity concentrations demonstrated a negative relationship between high salinity stress and P. longissimus survival, which only survived at low salinity concentrations and with a lower survival rate observed when compared to P. litoralis. We suggest that P. litoralis has a higher tolerance to salinity fluctuations than P. longissimus and that both species of littoral earthworms prefer to inhabit the ecotone between terrestrial and marine habitats. Our results support the current distribution patterns of these two littoral earthworm species, where P. litoralis is a cosmopolitan species and occurs in a very wide range of subtemperate and tropical coastal ecosystems 4,6,7,21-24 with dispersal mechanisms including transportation by wooden vessels 5,25 . Salinity fluctuation is a principal environmental factor affecting the distribution of marine and estuarine species 26 and high euryhalinity may be a factor in its global distribution similar to the case of the marine intertidal gastropod Stramonita brasiliensis 27 . In contrast, a poor tolerance to salinity changes is a factor that limits the dispersal ability of P. longissimus, concordant with it only being reported in the coastline of Thailand and Peninsular Malaysia 6,8 .
The effects of salinity have also been reported in other earthworms, where it affected the quality of products in composting, limited the growth of earthworms, and reduced the weight and survival percentage of earthworms 12,28-30 . Here, the salinity was found to affect the weight change of both littoral earthworm species. The highest level of weight gain in P. litoralis was observed at 12 h in all salinity concentrations, which we suggest supports the notion that water enters by osmosis increasing the worm's weight but this response becomes limited over time. The result showed that P. litoralis was able to resist salinity changes and match the salinity fluctuations in their natural microhabitat as previously reported in P. bermudensis (which is now considered a synonym of P. litoralis) 14 , with a salinity concentration of 20-30 ppt being the optimal range for P. litoralis.
Understanding the fundamental knowledge of osmotic strategies in gaining salinity tolerance should provide insights into the evolutionary processes of physiological adaptation to saline waters and habitat selection strategies. Several studies have been reported on osmoregulation in earthworms 28,31,32 . Since, osmoregulation is very important in estuarine intertidal species, we investigated how the coelomic fluid osmolality of these two Pontodrilus increases with increasing medium salinity to evaluate how they the species handle salinity fluctuations. Changes in salinity concentrations will cause a change in the osmotic pressure of an organism through osmoregulation processes. The body fluid osmolarity changed in accord with the outside exposure medium. These findings are in agreement with the present hypothesis and confirm that P. litoralis and P. longissimus are osmoconformers with regard to salinity change [33][34][35] . In conclusion, the current study examined the effects of different salinities on the two species of earthworms in the genus Pontodrilus. A high salinity concentration www.nature.com/scientificreports/ resulted in a reduced weight and survival percentage of the cosmopolitan earthworm P. litoralis and was fatal to the endemic P. longissimus. The osmoregulation ability of these two different Pontodrilus species of littoral earthworms to changes in salinity appear to explain their distribution pattern and habitat preference throughout Thailand's coastal areas, where P. litoralis has a higher tolerance to salinity fluctuations than P. longissimus corresponding with its greater ability to disperse throughout the world. Habitat patterns associated with salinity indicated that their natural habits are based upon the least osmolarity stressful environment. Theoretically, a specific adaptation is required, they either control their body fluid equivalent to the composition of the sea or actively conserve or shed excess salts.

Material and methods
Field sampling and observation. Littoral earthworm genus Pontodrilus were surveyed throughout the coastal areas of Thailand, both on the Gulf of Thailand (east coast) and the Andaman Sea (west coast) from January 2015 to January 2016. The sampling sites are shown in Fig. 1. During the field collection, geographic variation between the sampling sites was observed and photographed (type of biotope), with salinity of the sampling site were recorded.
Laboratory experiment. Test organism. Adult P. litoralis (weight 190.31 ± 30.21 mg) and P. longissimus (weight 532.49 ± 146.17 mg) with a well-developed clitellum were collected from Hat Bang Saen, Mueang Chonburi, Chonburi, Thailand and from Hat Pakmeng, Sikao, Trang, Thailand, respectively. Only active worms were acclimated to laboratory conditions. The earthworms were rinsed with distilled water and left for a day on wet paper, moistened with artificial seawater without soil to avoid their stomach contents.
Test substances and medium. The sand was transported back from the field to the laboratory and oven-dried for 24 h. Different salinities of the test medium (0, 10, 20, 30, 40 and 50 ppt) were prepared by adding artificial sea salt to distilled water, and the salinity was then adjusted accordingly using a refractometer (ATAGO, Master-S10M).
Experimental procedure. Long-term (28 d) salinity exposure. A similar working procedure was applied to the long-term exposure (28 d) where the earthworms were exposed to different salinities (0, 10, 20, 30, 40 and 50 ppt) in the presence of the sand as a substrate. Round plastic containers (internal diameter 170 mm, height 130 mm) were filled with 2 kg of sand. Each container had an inlet and an outlet to allow for running water and cleaning processes and was lined with a paper bag in order to prevent the worms from escaping out of the container. Ten earthworms were released into each container with four replicates of each treatment. The tests were conducted at ambient temperature (30 ± 2 °C) and a 16:8 h light:dark cycle (light intensity of 400-800 lx). Finely ground cow dung (30 g) was added each week to each container as food 36 . At the end of the exposure, the survival rate and weight change of each treatment were calculated.
Short-term (0-96 h) salinity exposure. The short-term exposure test was conducted in a round, flat-bottomed (internal diameter 48 mm, height 80 mm) container. Each test container was filled with 20 ml of the prepared medium with the designated salinity level (artificial seawater at 0, 10, 20, 30, 40 and 50 ppt) and then exposed for different duration (3, 6, 12, 24, 48, 72 and 96 h). Each treatment was performed with three replicates (one individual per replicate). The test units were closed with a perforated plastic film to prevent the earthworms from escaping. The experiment was carried out at ambient room temperature (25 ± 2 °C) with a 16:8 h light:dark illumination cycle (light intensity of 400-800 lx). The working medium was checked and changed every day in order to avoid salinity changes due to evaporation. The behavior of the earthworms was recorded using a Canon 650D camera. The earthworms were weighed after 0, 3, 6, 12, 24, 48, 72 and 96 h of exposure, and the change in weight was calculated. The earthworms were prepared for osmolality testing.
Data analysis. One-way ANOVA was used to evaluate differences among different treatments on the assessed parameters, while Tukey's post hoc test was performed to test for the significance of any differences between the means of different treatments. All statistical analyses were performed using the Minitab 16 Statistical software and significance was accepted at P < 0.05 level.
Determination of the body (coelomic) fluid osmolarity. Earthworms from the short-term exposure experiment were used for osmolality determination. Their coelomic fluid (≥ 20 μL) was extracted from the coelomic cavity with the aid of a thin glass capillary. A Micro Osmometer (Fiske Model 210 Micro Osmometer) was used to determine the osmolality of each solution using freezing point depression and was calibrated using the manufacturer-supplied standards.

Data availability
The data that support the findings of this study are openly available in Zenodo at https:// doi. org/ 10. 5281/ zenodo.